At present, “abnormal structural proteins” have drawn attention as common mechanisms of developing many neurodegenerative diseases that develop with aging, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, and prion disease, and molecular nature of such proteins has been studied. Deposition of two types of fibrous aggregates in the brain: i.e., deposition of senile plaque primarily composed of amyloid β proteins (Aβ) (Selkoe, D. J., Annu. Rev. Neurosci., 12, 463-490, (1989); and Glenner, G. G. and Wong, C. W., Biochem. Biophys. Res. Commun., 120 (3), 885-890, (1984)); and deposition of neurofibrillary degeneration (paired helical filament (PHF)) primarily composed of phosphorylated tau proteins (Ihara, Y. et al., J. Biochem., 99, 1807-1810, (1986); and Grundke-Iqbal, I. et al., Proc. Natl. Acad. Sci. U.S.A., 83, 4913-4917, (1986)) have been reported as the pathological features of Alzheimer's disease. In recent study of Alzheimer's disease that is considered to be caused by a plurality of various pathogens, amyloid β protein deposition has become considered to be a common pathway for the development of all such diseases. Amyloid β protein is a peptide that is cleaved as a molecular species consisting of 40 (Aβ1-40) or 42 (Aβ1-42) residues from its precursor substance (i.e., amyloid precursor protein (APP)), and generation and decomposition thereof advance while maintaining homeostasis in normal humans. Excessive deposition of amyloid β proteins in Alzheimer's disease, however, is considered to result from deregulation during cleavage or decomposition. In this description, the former proteins (Aβ1-40) may be referred to as “amyloid β40,” “amyloid β40 monomers,” or “monomeric amyloid β40 proteins,” and the latter proteins (Aβ1-42) may be referred to as “amyloid β42,” “amyloid β42 monomers,” or “monomeric amyloid β42 proteins.” A minor amount of amyloid β proteins is cleaved as a molecular species consisting of 43 (Aβ1-43) residues, and such proteins may be referred to as “amyloid β43,” “amyloid β43 monomers,” or “monomeric amyloid β43 proteins.”
The deposited amyloid β proteins act on neurons as neurotoxins and cause synaptic degeneration and subsequent neuronal cell death. This mechanism is considered to cause selective neuronal drop out, which may cause progressive dementia of Alzheimer's disease. Also, it has been reported that amyloid β proteins do not exhibit neuronal cell death activity when they were released extracellularly as water-soluble peptides (hereafter the term “neuronal cell death activity” may be referred to as “toxicity”) and that amyloid β proteins self aggregate and form amyloid β fibers, upon which they acquire toxicity (Lorenzo, A. and Yankner, B. A., Proc. Natl. Acad. Sci. U.S.A., 91, 12243-12247, (1994)). When a solution containing toxic amyloid β protein that contains amyloid β fibers is added to cultured neurons at high concentration, such neurons are known to be led to death. Accordingly, the amyloid β fibers were considered to be the entity to induce neuronal cell death in Alzheimer's disease.
Thus, an experimental system wherein neuronal cells are induced to die with the addition of toxic amyloid βproteins containing amyloid β fibers has been considered to reflect the neuronal cell death in Alzheimer's disease and has often been employed in screening for inhibitors of neuronal cell death or the like. In recent years, however, the following facts have been reported, which would suggest that the toxic entity of the amyloid β protein is not the amyloid β fiber. That is, (1) the concentration of amyloid β fibers in a toxic amyloid protein-containing solution necessary for inducing neuronal cell death is several tens of μM (Yankner, B. A., et al., Science, 250, 279-282, (1990)), which is 1,000 times or greater than that in the brain of an Alzheimer's patient; (2) the amount of amyloid β fibers deposited in the brain of an Alzheimer's patient is not always correlated with the impairment of higher-order functions, such as memory or cognitive function, and no clinical symptom may be developed even though a large quantity of amyloid βfibers are deposited; (3) the site of amyloid β deposition is not always consistent with the site of neuronal drop out in the brain; (4) abnormality is observed in learned behavior before or without the deposition of amyloid β fibers in the brain of an APP-overexpressing mouse; and (5) increase in the water-soluble amyloid β protein content in the brain of an Alzheimer's patient occurs 10 or more years ahead of the deposition thereof.
The present inventors had proposed a solution containing highly toxic self-aggregated amyloid β proteins that would induce neuronal cell death at a concentration equivalent to that of the self-aggregated amyloid β proteins that exist in the bodies of Alzheimer's patients or other diseases and a method for producing such solution (JP Patent Publication (Kokai) No. 2001-247600 A). The present inventors had also discovered a method for isolating neurotoxins contained in the aforementioned solution containing self-aggregated amyloid β proteins and analyzed the neurotoxins. As a result, such neurotoxins were found to be self-aggregated amyloid β proteins in the form of particles having diameters of approximately 10 nm to 20 nm, and these particles were designated as amylospheroids. In accordance with such designation, self-aggregated amyloid β proteins in the form of particles having diameters of approximately 10 nm to 20 nm may be referred to as “amylospheroid” herein.
Amylospheroid induces neuronal cell death at a concentration equivalent to that of amyloid β proteins that exist in the brain of an Alzheimer's patient, and phosphorylates a tau protein, which is another pathological marker in the process where amylospheroid causes nerves to die. Since these mechanisms are consistent with the pathological conditions of Alzheimer's disease, amylospheroid was considered to be the toxin of the amyloid β protein in the brain. If (1) an antibody that inhibits amylospheroid formation or (2) an antibody that inhibits toxicity of amylospheroid against neuronal cells is obtained, accordingly, such antibody can be used for a therapeutic or preventive agent for Alzheimer's disease. If (3) an antibody having greater reactivity with amylospheroid than with amyloid β monomers or amyloid β fibers is obtained, such antibody can be utilized in the assay for diagnosing Alzheimer's disease.
A method for preparing an antibody that reacts with an amylospheroid antigen has already been known. However, antibodies having the aforementioned properties (1) to (3) had not yet been obtained for the following reasons. That is, (a) amylospheroid is an aggregate of amyloid β proteins, and it is generally difficult to obtain an antibody reacting with aggregated proteins, (b) the mechanism of amylospheroid aggregation or the correlation between the structure and the function is not clear, and essential antigen portions necessary for the functions (1) and (2) of the antibody are not clear. Accordingly, it is particularly difficult to obtain an antibody that reacts specifically with amylospheroid and inhibits toxicity of the protein against neuronal cells.